The long-term objective of our research is to understand how pain-sensing neurons function at the molecular level under both normal conditions and chronic pain states. In mammals, the initial detection of pain stimuli is carried out by a group of sensory neurons, known as nociceptive neurons or pain-sensing neurons. Under chronic pain states, nociceptive neurons become hypersensitive to pain stimuli. Recently, we have identified a large family of G protein coupled receptors, called Mrgs, expressed specifically in pain-sensing neurons. We also found several RFamide peptides, including FMRFamide, gamma2-MSH and NPFF, acting as ligands for Mrgs in heterologous systems. Our electrophysiological studies indicate that FMRFamide can significantly increase excitability of nociceptive neurons in an Mrg-dependent manner. Our behavioral analysis showed that Mrg gene cluster knockout mice exhibit a prolonged inflammatory pain response as compared to wild-type mice. These data suggest that Mrgs play a modulatory role in pain. Here we propose to study the function of Mrgs in pain sensation further using Mrg knockout mice. Aim I is to analyze pain behavioral phenotypes in Mrg-deficient mice, from which we would like to determine whether Mrgs act to promote or reduce pain sensitivity in vivo and whether they play roles in chronic pain. In Aim II, we will characterize whether the injection of Mrg ligand peptides into wild-type mice can cause nociceptive effects. If they do, we will determine whether such effects are Mrg-dependent. Our recent data suggest that mast cells are likely to express the endogenous ligands for Mrgs. One promising candidate is the mammalian peptide NPFF. We will test whether mast cells express the peptide. If they do, we will check whether the degranulation of mast cells can release the peptide. We will also investigate Mrg receptor binding sites by autoradiography using radiolabeled NPFF and gamma2-MSH. Aim III is to study the molecular mechanisms of neuronal hyperexcitability induced by Mrg ligand peptides. Many ion channels have been implicated in regulating neuronal excitability of nociceptive neurons. We will employ electrophysiological recording techniques to determine whether Mrgs can modulate the activities of the ion channels in cultured DRG neurons. The proposed research will enhance our understanding of Mrg function in pain, which may allow the development of specific novel therapeutic agents for chronic pain with limited side effects.